Expression of T cell surface proteins fused to fluorescent proteins enables investigation of the formation of the immunological synapse in living cells. Using fluorescence resonance energy transfer (FRET) between these proteins, interactions between molecules during recognition of antigen can be visualized, since FRET requires that the proteins be within 10nm of each other. CD3zeta fused to cyan fluorescent protein (CFP), a derivative of green (G)FP, and CD4 fused to yellow (Y)FP will be used to analyze the immunological synapse on class II-restricted T cells. CFP and YFP form a suitable pair for FRET, which enables measurement of TCR-CD4 interactions in living cells. Preliminary data show FRET between these molecules on T cells recognizing antigen. In addition to transfecting the genes into T cell lines, transgenic mice will be made that express these proteins, to provide a source of naive cells useful for many experiments. The earliest time points in antigen recognition, before formation of the mature synapse, will be studied, to compare CD4 and TCR recruitment to the contact region, and their interaction, to Ca2+ signaling. Synapse formation in response to antagonists and during antagonism of a response to antigen will also be investigated. The role of lipid rafts will be probed using raft-associated FPs. The ability of T cells to make synapses with naturally processed antigen on dendritic cells will be investigated. There is evidence from crystallography that CD4 dimerizes. 1 ALLERGY AND IMMUNOLOGY STUDY SECTION 3 R01GM65230-01 OCTOBER 2001 GASCOIGNE, NICHOLAS R. This will be tested using FRET between CD4-YFP and CD4-CFP. Fine synapse-related "tether" structures have been observed, and their relevance to T cell activation will be studied. Little is known about the immunological synapse in class I-restricted T cells. CD3zeta-CFP and CD8-YFP will be used to investigate formation of the synapse and interactions between the TCR and coreceptor in a class I-restricted system. A T cell receptor (TCR) system where agonist and antagonist peptides are particularly well characterized and where effects on TCR-co-receptor interaction in the synapse can be related to the binding constants for the TCR-MHCp complex will be used. The role of the "TCR alpha-chain connecting peptide motif" (alpha-CPM) in the synapse will be investigated. Using T cells from mice transgenic for CD3-CFP, co-receptor-YFP, and TCR, differences in synapse formation between naive, activated and memory T cells will be investigated. Two-photon microscopy will be used to image T cells interacting with tissue cells within lymph node or thymus. Immunological synapse formation during an immune response in lymph node and during positive and negative selection in the thymus will be visualized.